Dendritic spines are the main receipts of excitatory synapses in the central nervous system. One of the main hypothesis trying to explain the physiological role of dendritic spines claims that these small dendritic structures form a biochemical compartment for intracellular calcium. According to this hypothesis activation of synaptic inputs innervating a spine induced a localized increase of the inti-acellular calcium concentration in the spine mediated by influx of calcium via NMDA receptor channels or metabotropic glutamate receptor mediated release of calcium from intracellular stores. The synaptic induced spine calcium transients are thought to mediate long term changes in synaptic efficacy such as long term potentiation and depression, which may play a key role in m-emory and learning. Previously no intracellular biological active substance was uncaged by a two-photon laser in brain slices. Presently we are in the final stages of setting up a system which will allow simultaneous two photon uncaging and imaging in brain slices. With the use of this set up we plan to perform several experiments addressing the role of dendritic spines as calcium compartments, and their possible role in long term synaptic plasticity. 1) Uncaging of calcium in dendritic spines and neighboring parent dendrites. In this project we will characterize the diffusion of calcium into and from the spines, and thus examine the role of dendritic spines as localized calcium compartments. Recently the diffusion of uncaged fluorescein was examined in dendritic spines, but the relevance of these experiments to intracellular calcium in dendritic spines is unclear. 2) Uncaging of IP3 in dendritic spines and neighboring parent dendrites and characterization of the calcium transients resulting from IP3 mediated release of calcium from intracellular stores.